Leo Kurian

Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs, progerin and its ageing-associated phenotypic consequences are restored. Specifically, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs, also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing.

Conversion of human fibroblasts to angioblast-like progenitor cells

Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct, in the absence of proliferation and multipotent progenitor generation, or indirect, by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state, induced by brief exposure to reprogramming factors, followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells, including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies.

In Vivo Activation of a Conserved MicroRNA Program Induces Mammalian Heart Regeneration

Heart failure is a leading cause of mortality and morbidity in the developed world, partly because mammals lack the ability to regenerate heart tissue. Whether this is due to evolutionary loss of regenerative mechanisms present in other organisms or to an inability to activate such mechanisms is currently unclear. Here we decipher mechanisms underlying heart regeneration in adult zebrafish and show that the molecular regulators of this response are conserved in mammals. We identified miR-99/100 and Let-7a/c and their protein targets smarca5 and fntb as critical regulators of cardiomyocyte dedifferentiation and heart regeneration in zebrafish. Although human and murine adult cardiomyocytes fail to elicit an endogenous regenerative response after myocardial infarction, we show that in vivo manipulation of this molecular machinery in mice results in cardiomyocyte dedifferentiation and improved heart functionality after injury. These data provide a proof of concept for identifying and activating conserved molecular programs to regenerate the damaged heart.

Identification of Novel Long Noncoding RNAs Underlying Vertebrate Cardiovascular Development

Background— Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development. Methods and Results— By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs, TERMINATOR, ALIEN, and PUNISHER, specifically expressed in undifferentiated pluripotent stem cells, cardiovascular progenitors, and differentiated endothelial cells, respectively. Functional characterization, including localization studies, dynamic expression analyses, epigenetic modification monitoring, and knockdown experiments in lower vertebrates, as well as murine embryos and human cells, confirmed a critical role for each lncRNA specific for each analyzed developmental stage. Conclusions— We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development, and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development, mesodermal commitment, and cardiovascular specification.

Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA

Polyamines are essential organic polycations with multiple cellular functions relevant for cell division, cancer and ageing. Regulation of polyamine synthesis is mainly achieved by controlling the activity of ornithine decarboxylase (ODC) through an unusual mechanism involving ODC antizyme, the binding of which disrupts homodimeric ODC and targets it for ubiquitin-independent degradation by the 26S proteasome. Whereas mammals express several antizyme genes, we have identified a single orthologue, termed OAZ1, in Saccharomyces cerevisiae. Similar to its mammalian counterparts, OAZ1 synthesis is induced with rising intracellular polyamine concentrations, which also inhibit ubiquitin-dependent degradation of the OAZ1 protein. Together, these mechanisms contribute to a homeostatic feedback regulation of polyamines. Antizyme synthesis involves a conserved +1 ribosomal frameshifting (RFS) event at an internal STOP codon during decoding of its messenger RNA. Here we used S. cerevisiae OAZ1 to dissect the enigmatic mechanism underlying polyamine regulation of RFS. In contrast with previous assumptions, we report here that the nascent antizyme polypeptide is the relevant polyamine sensor that operates in cis to negatively regulate upstream RFS on the polysomes, where its own mRNA is being translated. At low polyamine levels, the emerging antizyme polypeptide inhibits completion of its synthesis causing a ribosome pile-up on antizyme mRNA, whereas polyamine binding to nascent antizyme promotes completion of its synthesis. Thus, our study reveals a novel autoregulatory mechanism, in which binding of a small metabolite to a nascent sensor protein stimulates the latter’s synthesis co-translationally.

Conversion of Human Fibroblasts Into Monocyte‐Like Progenitor Cells

Reprogramming technologies have emerged as a promising approach for future regenerative medicine. Here, we report on the establishment of a novel methodology allowing for the conversion of human fibroblasts into hematopoietic progenitor‐like cells with macrophage differentiation potential. SOX2 overexpression in human fibroblasts, a gene found to be upregulated during hematopoietic reconstitution in mice, induced the rapid appearance of CD34+ cells with a concomitant upregulation of mesoderm‐related markers. Profiling of cord blood hematopoietic progenitor cell populations identified miR‐125b as a factor facilitating commitment of SOX2‐generated CD34+ cells to immature hematopoietic‐like progenitor cells with grafting potential. Further differentiation toward the monocytic lineage resulted in the appearance of CD14+ cells with functional phagocytic capacity. In vivo transplantation of SOX2/miR‐125b‐generated CD34+ cells facilitated the maturation of the engrafted cells toward CD45+ cells and ultimately the monocytic/macrophage lineage. Altogether, our results indicate that strategies combining lineage conversion and further lineage specification by in vivo or in vitro approaches could help to circumvent long‐standing obstacles for the reprogramming of human cells into hematopoietic cells with clinical potential. Stem Cells 2014;32:2923–2938

A lncRNA Perspective into (Re)Building the Heart

Our conception of the human genome, long focused on the 2% that codes for proteins, has profoundly changed since its first draft assembly in 2001. Since then, an unanticipatedly expansive functionality and convolution has been attributed to the majority of the genome that is transcribed in a cell-type/context-specific manner into transcripts with no apparent protein coding ability. While the majority of these transcripts, currently annotated as long non-coding RNAs (lncRNAs), are functionally uncharacterized, their prominent role in embryonic development and tissue homeostasis, especially in the context of the heart, is emerging. In this review, we summarize and discuss the latest advances in understanding the relevance of lncRNAs in (re)building the heart.

A post-transcriptional program coordinated by CSDE1 prevents intrinsic neural differentiation of human embryonic stem cells

While the transcriptional network of human embryonic stem cells (hESCs) has been extensively studied, relatively little is known about how post-transcriptional modulations determine hESC function. RNA-binding proteins play central roles in RNA regulation, including translation and turnover. Here we show that the RNA-binding protein CSDE1 (cold shock domain containing E1) is highly expressed in hESCs to maintain their undifferentiated state and prevent default neural fate. Notably, loss of CSDE1 accelerates neural differentiation and potentiates neurogenesis. Conversely, ectopic expression of CSDE1 impairs neural differentiation. We find that CSDE1 post-transcriptionally modulates core components of multiple regulatory nodes of hESC identity, neuroectoderm commitment and neurogenesis. Among these key pro-neural/neuronal factors, CSDE1 binds fatty acid binding protein 7 (FABP7) and vimentin (VIM) mRNAs, as well as transcripts involved in neuron projection development regulating their stability and translation. Thus, our results uncover CSDE1 as a central post-transcriptional regulator of hESC identity and neurogenesis.Unlike transcriptional regulation of hESC identity, little is known post-transcriptionally. Here, the authors show that the RNA binding protein CSDE1 regulates core components of hESC identity, neurectoderm commitment and neurogenesis to maintain pluripotency and prevent neural differentiation.

Sense-Antisense lncRNA Pair Encoded by Locus 6p22.3 Determines Neuroblastoma Susceptibility via the USP36-CHD7-SOX9 Regulatory Axis

Trait-associated loci often map to genomic regions encoding long noncoding RNAs (lncRNAs), but the role of these lncRNAs in disease etiology is largely unexplored. We show that a pair of sense/antisense lncRNA (6p22lncRNAs) encoded by CASC15 and NBAT1 located at the neuroblastoma (NB) risk-associated 6p22.3 locus are tumor suppressors and show reduced expression in high-risk NBs. Loss of functional synergy between 6p22lncRNAs results in an undifferentiated state that is maintained by a gene-regulatory network, including SOX9 located on 17q, a region frequently gained in NB. 6p22lncRNAs regulate SOX9 expression by controlling CHD7 stability via modulating the cellular localization of USP36, encoded by another 17q gene. This regulatory nexus between 6p22.3 and 17q regions may lead to potential NB treatment strategies.

Co-translational Polyamine Sensing Co-translational polyamine sensing by Nascent ODC Antizyme ODC antizyme

Polyamines are essential biogenic poly-cations with important roles in many cellular processes, including translation and DNA replication. High levels of polyamines have been linked to cancer. Increased levels of polyamines, however, have also been shown to promote longevity. Cellular regulation of polyamines involves a variety of complex mechanisms that regulate uptake and excretion as well as synthesis and catabolism of polyamines. A key enzyme in the biosynthesis of polyamines is ornithine decarboxylase (ODC). The activity and stability of this homodimeric protein is controlled by ODC antizyme (OAZ). OAZ binds ODC monomers and thereby targets them for ubiquitin-independent degradation by the proteasome. OAZ is encoded by an interrupted open reading frame. As a consequence, synthesis of the functional protein is slowed and only occurs when a ribosomal frameshift (RFS) event takes place. High polyamine concentrations lead to an increased efficiency of translation of OAZ mRNA. Our studies have revealed that nascent OAZ polypeptide causes a translational arrest of ribosomes when polyamine concentration is low. At higher concentrations, binding of polyamines to nascent OAZ prevents stalling of the ribosomes on OAZ mRNA and thereby promotes synthesis of full-length ODC antizyme. Co-translational sensing of polyamines thereby contributes to a homeostatic regulation of polyamine biosynthesis.